PROJECT SUMMARY/ABSTRACT Defects in the assembly and maintenance of mitochondrial oxidative phosphorylation (OXPHOS) machinery lead to a range of degenerative illnesses, including diabetes, cancer, and neurodegenerative diseases. OXPHOS complexes are encoded on both the nuclear and mitochondrial genomes, so their biogenesis requires the precise coordination of gene regulatory mechanisms across genomes. To this end, mitochondrial biogenesis and stress response programs involve the simultaneous transcriptional upregulation of nuclear- encoded OXPHOS genes and mitochondrial gene expression factors. These transcriptional programs are thought to facilitate nuclear-mitochondrial balance, where mitochondrial-encoded OXPHOS subunits assemble in stoichiometric ratios with their nuclear-encoded counterparts. We recently observed in Saccharomyces cerevisiae that transcription regulation of nuclear-encoded and mitochondrial-encoded OXPHOS subunits are not coordinated during carbon source adaptation. Instead, the cell synchronizes the translational regulation of OXPHOS subunits across compartments. Whether synchronized translation is a widespread response remains unknown. The goal of this proposal is to determine how mitochondrial and nuclear genomes are co-regulated, particularly during protein synthesis, throughout mitochondrial biogenesis and stress response programs. As an extension of our recent work in yeast, Aim 1 will investigate whether synchronous translation programs occur across a range of environmental and mitochondrial stress adaptation programs. We will also determine how the synchronous translation regulation occurs by determining the role of key mitochondrial translation regulators in the dynamic regulation of OXPHOS genes during carbon source adaptation. This will be done using our mitochondrial ribosome profiling approach and cytosolic ribosome profiling to measure protein synthesis and RNA-seq to measure global transcription. In Aims 2 and 3, we extend our studies to human cells through re-engineering ribosome profiling to robustly capture human mitochondrial translation. To test the approach, we will investigate how a putative translation activator, TACO1, impacts human mitochondrial translation. We will investigate nuclear-mitochondrial co-regulation after acute mitochondrial stress induced by chemical inhibition of OXPHOS complexes, mimicking OXPHOS dysfunction in disease processes. Finally, we will investigate mitochondrial and nuclear gene expression programs during the in vitro differentiation of iPS cells to cardiomyocytes, when extensive mitochondrial biogenesis occurs. Determining the regulation and the extent of nuclear-mitochondrial co-regulation will provide critical insight towards understanding how imbalanced production of OXPHOS subunits transpires in disease states.